1. Field of the Invention
The present invention relates to an agent for photodynamic therapy (PDT) containing porous silicon (PSi) and the method of quantitative measurement of reactive oxygen species (ROS) produced therefrom. Specifically, the present invention relates to an agent for PDT containing PSi that generates heat sufficient to kill cancer cells with generating little ROS and the method of quantitative measurement of a very small amount of ROS produced therefrom through X-ray diffraction (XRD) analysis.
2. Description of the Related Art
All of the current cancer treatments, such as radiation therapy, chemotherapy etc. attack rapidly proliferating cells. Unfortunately, these treatments attack not only cancer cells but also some of normal cells. As a result, the above treatments can deteriorate natural anti-tumor defense mechanism, as well as can cause fatal side effects. For example, radiation therapy or chemotherapy impairs rapidly dividing cells in the immune system and suppress anti-tumor and anti-infection response.
Besides the above side effects, the current cancer treatments do not satisfy the effect to be desired because they are lack of ability to selectively attack cancer cells. As a result, radiation therapy, chemotherapy or combination thereof cannot treat cancer effectively. Currently, the principal cancer treatment is the surgical removal of cancer cells. The surgical method is often performed together with radiation therapy and chemotherapy, and surgical amputation and treatments with high toxicity should be used in order to destroy all cancer cells in the real situation.
Photodynamic Therapy (PDT) was developed as a part of the efforts to minimize the above side effects of cancer treatments and improve total efficacy. PDT comprises administrating a photosensitizer to a human body for localization focusing on cancer cells and irradiating light having specific and appropriate wavelength to the cancer cells containing the photosensitizer. Accordingly, PDT can generate therapeutic response on specific human tissues such as cancer through a combination of a photosensitizer and site-specific irradiation because it can apply active light having appropriate wavelength to a specific site.
The traditional PDT destroys cancer cells using ROS produced during irradiation to a photosensitizer. A photosensitizer for PDT is required to satisfy following conditions;                Firstly, a high quantum yield to produce ROS, secondly, long wavelength of absorbing light, and thirdly, low toxicity in a non-irradiated state.        
Therefore, aromatic molecules or dye molecules have been clinically employed as photosensitizers for the past few decades since they have potential to generate ROS. Also recently some nanomaterials, such as TiO2, ZnO, Au, CNT (carbon nanotube), PSi (porous silicon), fullerenes etc. have been reported as new photosensitizers that can generate ROS.
ROS means unstable chemical species such as singlet oxygen (1O2), superoxide anion (O2−), hydroxyl radical (.OH), etc. which can cause cells irreversible damage through photobiological activities. In addition, there have been many reports on side effects arising from ROS production as follows:
First of all, the short-term side effects of light exposure include swollen skin, red flecks and pains. Also other side effects including appetite loss and a sore throat on swallowing, etc. last for more than six weeks.
Secondly, ROS causes structural and functional damage in the long term by reacting with biological molecules such as deoxyribonucleic acid (DNA), proteins and lipids.
Thirdly, this type of oxidative damage accumulated in human bodies can cause diseases such as heart disease, cancer, etc. eventually.
Recently, a new PDT technique in which single-wall carbon nanotubes (SWCNT) are used as a cancer treatment agent has been reported. The CNT used in this new PDT can absorb near infrared ray (NIR) whose wavelength range is from 700 to 800 nm and the heat released from the CNT can destroy cancer cells effectively. That is, if CNT to which folic acid or antibody is attached is administered to the cells using techniques such as endocytosis, the CNT administrated moves places where cancer cells exist in order to look for them because cancer cells contain a large number of antibody receptors. The NIR irradiation from the outside of the body at the stage allows the CNT to absorb the NIR. The CNT becomes excited through absorbing NIR energy and releases energy in the form of heat that can destroy surrounding cancer cells. The difference between the PDT based on the present invention and the traditional PDT is that the former destroys cancer cells using heat released from a photosensitizer by irradiating the NIR while the latter PDT destroys cancer cells using ROS released from a photosensitizer by irradiating visible ray. The traditional PDT can destroy only cancer cells existing near skin because visible ray can penetrate to depths of a few millimeters from human skin, but the new PDT can effectively destroy cancer cells located deep in human bodies because the NIR can penetrate human bodies well. In the new PDT, higher heat emission efficiency is preferred, while smaller amount of ROS emission causing side effects is preferred.
Anyway, it is important to know the exact ROS emission efficiency regarding photosensitizers whether it is the traditional PDT or the new PDT.
Recent work by Yamakoshi et al. shows that other unstable chemical species such as O2− and .OH as well as 1O2 are also generated by light irradiation on a photosensitizer and they also destroy cancer cells. Therefore, it is necessary to measure emission efficiency for all ROS including 1O2, O2− and .OH. Various techniques have been developed to measure the amount of ROS for the past decades, but these techniques were mostly developed to assess the quantum yield only for 1O2 generation (Yoko Yamakoshi J. AM. CHEM. SOC. 125, 12803-12809 (2003); Carre, C. et al., J. Chim. Phys. Phys-Chim. Biol., 84: 577-85 (1987); Darmanyan. A. P., Chem. Physics. Lett., 91: 391-400 (1982); Chattopadhyay, S. K. et al., J. Photochem., 24: 1-9 (1984); Olmsted, J., III, J. Am. Chem. Soc. 102: 66-71 (1980); Rossbroich, G. et al., J. Photochem., 31: 37-48 (1985); Heihoff, K. et al., Photochem. Photobiol., 51:634-41 (1990); Garner, A. et al., Singlet Oxygen, Reactions with Orgnic Compounds and Polymers. B. Ranby and J. F. Rabek (eds.), John Wiley & Sons, New York, N.Y., 1976, p. 48-53.). The present invention relates to an analysis technique that can simultaneously measure the amount of ROS of every kind released from a photosensitizer upon light irradiation.
On the other hand, the following are theoretical background to measure ROS efficiently using XRD analysis:
The exact expression for the intensity of a single-phase powder specimen in an X-ray diffractometer is as follows.
                    I        =                                            (                                                                    I                    σ                                    ⁢                  A                  ⁢                                                                          ⁢                                      λ                    3                                                                    32                                      π                    ⁢                                                                                  ⁢                    r                                                              )                        ⁡                          [                                                                    (                                                                  π                        0                                                                    4                        π                                                              )                                    2                                ⁢                                                      e                    4                                                        m                    2                                                              ]                                ⁢                                    (                              1                                  v                  2                                            )                        ⁡                          [                                                                                        F                                                        2                                ⁢                                  p                  ⁡                                      (                                                                  1                        +                                                                              cos                            2                                                    ⁢                          2                          ⁢                                                                                                          ⁢                          θ                                                                                                                      sin                          2                                                ⁢                        θcos                        ⁢                                                                                                  ⁢                        θ                                                              )                                                              ]                                ⁢                      (                                          e                                                      -                    2                                    ⁢                  M                                                            2                μ                                      )                                              [                  Expression          ⁢                                          ⁢          1                ]            
In Expression 1, I=integrated intensity per unit length of diffraction line, I0=intensity of incident beam, A=cross-sectional area of incident beam, λ=wavelength of incident beam, r=radius of diffractometer circle, μ0=4π×10−7 m kg C−2, e=charge on electron (C), m=mass of electron (kg), v=volume of unit cell (m3), F=structure factor, p=multiplicity factor, θ=Bragg angle, e−2M=temperature factor, and μ=linear absorption coefficient (m−1) which enters as the absorption factor ½ μ.
                    K        =                              (                                                            I                  σ                                ⁢                A                ⁢                                                                  ⁢                                  λ                  3                                                            32                                  π                  ⁢                                                                          ⁢                  r                                                      )                    ⁡                      [                                                            (                                                            μ                      0                                                              4                      π                                                        )                                2                            ⁢                                                e                  4                                                  m                  2                                                      ]                                              [                  Expression          ⁢                                          ⁢          2                ]                                R        =                  (                                                    1                                  v                  2                                            ⁡                              [                                                                                                  F                                                              2                                    ⁢                                      p                    ⁡                                          (                                                                        1                          +                                                                                    cos                              2                                                        ⁢                            2                            ⁢                                                                                                                  ⁢                            θ                                                                                                                                sin                            2                                                    ⁢                          θ                          ⁢                                                                                                          ⁢                          cos                          ⁢                                                                                                          ⁢                          θ                                                                    )                                                                      ]                                      ⁢                          e                                                -                  2                                ⁢                M                                                                        [                  Expression          ⁢                                          ⁢          3                ]                                I        =                  KR                      2            ⁢            μ                                              [                  Expression          ⁢                                          ⁢          4                ]            
If we put K and R as in Expression 2 and Expression 3 respectively, then the diffracted intensity is given like Expression 4. If the XRD analysis test condition is fixed in the above Expression 4, K is a constant, R depends on the kind and crystallographic orientation of the diffracted substance, and μ is the absorption coefficient of the diffracted substance.
For the ith element of a multicomponent system composed of many elements, Expression 4 can be written as below.
                              I          i                =                                            KR              i                                      2              ⁢                                                          ⁢                              μ                m                                              ⁢                      C            i                                              [                  Expression          ⁢                                          ⁢          5                ]            
In Expression 5, Ci denotes the volume fraction of the i th element and μm the absorption coefficient of the multicomponent system. Since absorption is an atomic process, the multicomponent system can be regarded as slabs of each of pure elements as many as the number of the components, that is, a multilayer system. Similarly to Expression 4, for the ith layer of a multilayer system, Expression 3 can be expressed as below.
                              I          i                =                                            KR              i                                      2              ⁢                                                          ⁢                              μ                T                                              ⁢                      C            i                                              [                  Expression          ⁢                                          ⁢          6                ]            
In Expression 6, Ci denotes the volume fraction of the i th layer and μT the absorption coefficient of the total multilayer system.
It is possible to efficiently measure the amount of ROS released from a photosensitizer during exposure to the NIR irradiation using the above expressions and the values obtained by the XRD analysis measurement.
Hence, the inventors of the present invention identified that the emission of ROS is suppressed when PSi is exposed to the NIR while studying photosensitizers with suppressed ROS generation causing many of the side effects. They also completed the present invention through developing a reliable and reproducible method to measure the amount of ROS released from PSi or CNT using XRD analysis.